Within the projetc group we had transfered the methodological expertise from senior researchers to younger collegues; PI has established already strong collaboration with several Slovenian groups with complementary expertise: Laboratory for biotechnology - KI, Department for biomolecular sciences IJS, Institute of cell biology Faculty of Medicine UL and Laboratory for molecular neurobiology from Faculty of Medicine UL Young researchers - PhD students: PhD in preparation Maruša Bizjak (Rajh), mentor: Mojca Pavlin, co-mentor Sergej Pirkamjer, title: Effects of metformin on cancer cells in vitro for different nutrients availability Masters degree finished within the project: SEMIČ, Suzana. Vpliv modulatorjev energijske presnove na preživetje celic raka dojke in vitro = The effect of modulators of energy metabolism on survival of brest cancer cells in vitro : magistrski študijski program Laboratorijska biomedicina. Ljubljana: [S. Semič], 2018. VIII, 56 f., graf. prikazi. [COBISS.SI-ID 4457585] GLAVAČ, Klavdija. Vpliv mikrookolja na preživetje in migracijo melanomskih celic z različnim metastatskim potencialom : magistrsko delo = The impact of the microenvironment on the survival and migration of melanoma cells with different metastatic potential : M. Sc. thesis, (Biotehniška fakulteta, Oddelek za agronomijo, Magistrsko delo magistrskega študija - 2. stopnja Biotehnologija, 80). Ljubljana: [K. Glavač], 2016. XI, 62 f., ilustr. http://www.digitalna-knjiznica.bf.uni-lj.si/biotehnologija/du2_glavac_klavdija.pdf. [COBISS.SI-ID 8593017] KEŠAR, Urša. Vpliv izbranih dejavnikov na protitumorno delovanje metformina v celični kulturi rakavih celic : magistrsko delo = The effect of selected factors on anticancer action of metformin in cultured cancer cells : M. Sc. thesis, (Biotehniška fakulteta, Oddelek za agronomijo, Magistrsko delo magistrskega študija - 2. stopnja Biotehnologija, 76). Ljubljana: [U. Kešar], 2016. IX, 64 f., ilustr. http://www.digitalna-knjiznica.bf.uni-lj.si/biotehnologija/du2_kesar_ursa.pdf. [COBISS.SI-ID 8592761] ŠKORJA, Nives. Vpliv metformina na mitohondrijski potencial in presnovo laktata v celicah raka dojke : magistrsko delo : magistrski študij - 2. stopnja = Effect of metformin on mitochondrial potential and lactate production in breast cancer cells : M. Sc. Thesis : Master Study Programmes. Ljubljana: [N. Škorja], 2016. XIII, 69, [1] f., ilustr. http://www.digitalna-knjiznica.bf.uni-lj.si/biologija/du2_skorja_nives.pdf. [COBISS.SI-ID 4083279] BSC dipoma TURK, Nika. Izražanje modificiranega gena pfkA glive Aspergillus niger v kvasovki Saccharomyces cerevisiae : diplomsko delo, univerzitetni študij = Expression of a modified gene pfkA of fungi Aspergillus niger in yeast Saccharomyces cerevisiae : graduation thesis, university studies, (Biotehniška fakulteta, Enota medoddelčnega študija mikrobiologije, Ljubljana, Diplomske naloge, 541). Ljubljana: [N. Turk], 2015. XIII, 76 f., ilustr. http://www.digitalna-knjiznica.bf.uni-lj.si/mikrobiologija/dn_turk_nika.pdf. [COBISS.SI-ID 4558968] Several student research projects were published on conferences.
D.09 Tutoring for postgraduate students
COBISS.SI-ID: 8593017We used the yeast Saccharomyces cerevisiae as a model organism for studying deregulate glycolytic flow that was induced by the insertion of the gene encoding cancer specific shorter form of 6-phosphofructo-1-kinase (Pfk). It showed up that the transformant with the modified human Pfk enzyme can't grow on glucose due to unbalanced NADH/NADPH ratio caused by deregulated glycolysis. However, in a specific ecological niche growth condition were determined that allowed faster growth of the transformant with the modified, cancer characteristic Pfk enzyme in respect to the transformant with the native human Pfk enzyme. S. cerevisiae transformant encoding the highly active shorter Pfk fragments can be used as a model organism for studying deviant energetic metabolism in cancer cells (Warburg effect). RAMÓN GUTIÉRREZ CORROTO, Juan. Isolation and characterization of modified liver type 6-phosphofructo-l-kinase from tumorigenic and non tumorigenic cell lines : health biology degree : trabajo de fin de grado. Madrid: [J. Ramón Gutiérrez Corroto], 2016. 23 str., ilustr [COBISS.SI-ID 6106394] 6-Phosphofructo-1-kinase (PFK1) is an essential enzyme in the glycolitic pathway of all the cells, even more important is in the tumorigenic ones as they present Warburg effect and almost all the glucoses used are transformed into lactate. Kinetic characteristics of the enzyme are going to be measured from Jurkat cells and the expression of the shorter 47-49 kDa fragments are going to be tested with different aggressiveness tumor cells (MCF10A, MCF7 and Jurkat); these recently discovered fragments are made due to posttranslational modifications and are supposed to have different kinetics and inhibitions. GLADEK, Irena, KRISTL, Anja, LEŠNIK, Samo, LEGIŠA, Matic. Targeting modified 6-phosphofructo-1-kinase in cancer cells. V: Program of ISCAM2016, 3rd Annual Meeting of the International Society of Cancer Metabolism, 26-29 October 2016, Brussels - Belgium. Brussels: [s. n.]. 2016, str. 68. [COBISS.SI-ID 6021402]
F.04 Increase of the technological level
COBISS.SI-ID: 37878533At the 12th meeting of the Slovenian Biochemical Society, we presented the results obtained in the research for the master's thesis, which highlight the differences in the metabolic characteristics of breast and prostate cancer cells [1,3]. In addition, we showed that metformin increased the production of lactate despite the dichloroacetate (DCA)-stimulated oxidative metabolism in both cancer cell lines [3]. Using Western blot we demonstrated that DCA stimulated the oxidative metabolism by indirectly decreasing phosphorylation and thereby releasing the inhibition of the E1α subunit of the pyruvate dehydrogenase (PDHE1α), the key linking factor of the glycolytic and oxidative cell metabolism [3]. Despite metformin induced glycolysis in both cancer cell lines [1,2,3] it significantly reduced phosphorylation of PDHE1α [3], most probably indirectly via activation of AMPK [1,2,3]. Obtained results show similar metabolic responses of breast and prostate cancer cells after exposure to metformin and DCA, but it should be highlighted that the oxamate and DCA which inhibit glycolysis activated AMPK only in breast cancer cells, indicating breast cancer cells depend more on glycolysis than prostate cancer cells [3].
B.03 Paper at an international scientific conference
COBISS.SI-ID: 33406681The nutrient and metabolite availability likely plays an important role in orchestrating cancer-related alterations, including the steps involved in the metastatic cascade. Furthermore, nutrient availability can also alter effects of pharmacological compounds, such as metformin and 2-deoxy-glucose, that are used to target energy metabolism of cancer cells. Therea are numerous reports connecting nutrients availibility and energy metabolism linking to reposnse of cancer cells to different anticancer therapeutcis or to adjuvant treatmetn with modulatrs of cellular metabolism. However, molecular mechanisms linking alterations in energy metabolism to reduced or enhanced susceptibility of cancer cells to detach and to grow in anchorage independent conditions are not understood incompletely. Although cancer cells must detach to metastasize in vivo, the viability of floating cancer cells in vitro is rarely investigated. Here we show that co-treatment of anoikis-resistant MDA-MB-231 cells with metformin and 2-deoxy-D-glucose (2-DG) increased the percentage of floating cells, which were about 95% viable and resumed their proliferation once they were reseeded in the pharmacological compound-free medium. Similar effects on detachment were observed on anoikis-prone MCF-7 cells. Co-treatment of MDA-MB-231 cells with metformin and 2-DG induced a strong activation of AMP-activated protein kinase (AMPK), which was reduced by AMPK inhibitor compound C that prevented detachment of MDA-MB-231 cells. However, direct AMPK activators A-769662 and AICAR did not have any major effect on the percentage of floating MDA-MB-231 cells, indicating that AMPK activation is a necessary but not the main trigger required for detachment of cancer cells. Our results demonstrate that separate analysis of floating and attached cancer cells might be important for evaluation of anti-cancer agents. We further show that combined tretment with metformin and 2DG decreases rate of cell migration in standard transwell setup in agreement with reduced availability of energy for migration.
B.03 Paper at an international scientific conference
COBISS.SI-ID: 11606612In year 2015 we have established new laboratory within our department. Our facilities consist of i) fully equipped laboratory for synthesis of nanoparticles and ii) fully equipped cell culture laboratory. We have devloped several methods that enable us to analyse cancer cell metabolic pathways, different biochemical assays, cell migration, multicellular spheroids, co-cultures (e.g. HK2/UOK262), western blot. We have published also a methodological paper where flow cytometry, fluorescence microscopy and spectrofluorometry methods are compared. We have also developed a software for automatic cell counting. Methods available in our laboratories: •Synthesis of magnetic and gold nanoparticles, Synthesis of liposomes •Culturing of numerous mammalian cell lines and cell models, •Viability and proliferation assays (MTS, Propidium iodide, trypan blue exclusion, BrdU), •Reactive oxygen species measurements, •High-voltage electroporation and electrotransfection enabling various protocols for electroporation of cells in vitro and in vivo for RNAi/DNA/small molecules electrotransfer, •Western blot analysis •Microscopy ČIBEJ, Uroš, LOJK, Jasna, ŠAJN, Luka, PAVLIN, Mojca. Learn123. Ljubljana: Faculty of Electrical Engineering: Faculty of Computer and Information Science, 2015. http://lalg.fri.uni-lj.si/learn123/. [COBISS.SI-ID 10950996] kategorija: SU (S) točke: 0.5, št. avtorjev: 4 ARLAŠ, David, LOJK, Jasna, ŠAJN, Luka, ČIBEJ, Uroš, PAVLIN, Mojca. CellCounter. Ljubljana: Faculty of Electrical Engineering: Faculty of Computer and Information Science, 2014. http://lalg.fri.uni-lj.si/CellCounter/. [COBISS.SI-ID 10950740]
D.02 Establishment of a research centre, laboratory, study course, association
COBISS.SI-ID: 10746708